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(11) | EP 1 445 319 A1 |
| (12) | EUROPEAN PATENT APPLICATION |
| published in accordance with Art. 158(3) EPC |
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| (54) | GENE OF ENZYME REACTIVATING DNA DAMAGED BY ULTRAVIOLET LIGHT USING VISIBLE LIGHT |
| (57) It is intended to contribute to the solution of environmental problems caused by
destroying the ozone layer and serious problems concerning the food resources in the
21 st century by isolating a photoreactivating enzyme from an ultraviolet light-tolerant
rice plant, thus acquiring information on the photoreactivation mechanism of the plant
which still remains unknown mostly, and constructing an ultraviolet light-tolerant
plant with the use of a cloned gene. Genes encoding a photoreactivating enzyme originating
in rice plant, in particular, a gene encoding a protein as specified in the following
(a) or (b): (a) a protein comprising the amino acid sequence represented by SEQ ID
NO:1; and (b) a protein comprising an amino acid sequence derived from the amino acid
sequence (a) by deletion, substitution or addition of one to several amino acids and
having a photoreactivating enzyme activity. |
Technical Field
[0001] The present invention relates to a gene encoding a photoreactivating enzyme repairing DNA damaged by ultraviolet light, using visible light and a method for giving and enhancing the resistance to ultraviolet light, using the gene.
Background of the Invention
[0002] One of the environmental concerns recently focused on is the destruction of the ozone layer. What influences are caused by the destruction on our life? The life of biological organisms on the earth is supported by sunlight, and simultaneously, hazardous ultraviolet light in sunlight constantly threatens the life. Ultraviolet light is a part of sunlight at a wavelength of 100 to 400 nm and is largely divided into three parts, namely UV-C at 100 to 290 nm, UV-B at 290 to 320 nm and UV-A at 320 to 400 nm. Specifically, ultraviolet light at a wavelength of 320 nm or less is absorbed in the ozone layer, while ultraviolet light at the other wavelengths, namely a part of UV-B and UV-A pour on the earth. Particularly, an ultraviolet light component at a wavelength close to the wavelength for DNA absorption at 260 nm, namely UV-B, causes a structural modification in the base regions of DNA. The modification includes two types of CPD and 6-4 adduct and both of them are dimers generated through a covalent crosslinking between two pyrimidine (Py) moieties adjacent to each other. When the modification generated in DNA by ultraviolet light is defined as 100 %, CPD occupies 70 to 80 % and the 6-4 adduct occupies 20 to 30 %. These two structures inhibit DNA replication and transcription to cause cellular death and mutagenesis. It is believed that the onset of skin cancer caused by bathing in strong sunlight is triggered by these damages generated by ultraviolet light.
[0003] Biological organisms have various repair mechanisms for such damages. Therefore, biological organisms on the earth are not readily develop cancer even when they bath in sunlight. One of the mechanisms is photoreactivation. The repair system allows a photoreactivating enzyme to carry out the reverse reaction to that of Py + Py → CPD caused by ultraviolet light, using the energy of near ultraviolet light and blue light irradiated following ultraviolet light to return the CPD and 6-4 adduct generated by ultraviolet light to former state (1; Fig. 1). As the photoreactivating enzyme carrying out the repair, two types of enzyme exist; one specifically repairs CPD, while the other specifically repairs the 6-4 adduct (2,3). The presence of the CPD photoreactivating enzyme is confirmed widely among prokaryotic organisms and higher eukaryotic organisms. Once the ozone layer is destructed, the dose of ultraviolet light reaching the earth increases. Therefore, it is anticipated that more damages occur in DNA more than ever, leading to the limit of the repair, so that the biological organisms may be influenced by serious harms.
[0004] It is true with plants. Biological organisms with no direct need of sunlight can survive while avoiding sunlight, even if the dose of ultraviolet light increases. However, plants getting most of energy via photosynthesis cannot evade sunlight. Consequently, it is estimated that the influence of the destruction of the ozone layer on plants may be more serious than on biological organisms with no direct need of sunlight.
[0005] An experiment is reported recently, where Arabidopsis thaliana and Nicotiana were grown in environment at a higher ultraviolet light dose based on a possible decrease of the ozone layer in future as estimated from the current basal value of the dose of ultraviolet light (4). In other words, actual influences on plants were observed in a potential status assumed on the basis of the destruction of the ozone layer. The results are as follows. First, the dose elevation increases the cellular CPD and 6-4 adducts from the current levels, so that their growth is suppressed and their genes are increasingly recombined, leading to the elevation of the instability of the genomes, which involves the increase of the instability in the course of generations. In other words, the elevated ultraviolet light not only influences the generation itself but also gives such influences over some future generations. When the dose irradiated is retained, further, more mutations accumulate in a later generation, so that the generation turns more sensitive to ultraviolet light than preceding generations. This is due to the fact that Arabidopsis thaliana or Nicotiana is more influenced by ultraviolet light because Arabidopsis thaliana or Nicotiana is exposed to the external atmosphere during the term from the dehiscence of the anther as a reproductive organism to the stage of pollination with the pollen of Arabidopsis thaliana or Nicotiana. This is the case with most of plants on the earth. It may be considered that those described about Arabidopsis thaliana and Nicotiana can be induced by the destruction of the ozone layer and the subsequent increase of the dose of ultraviolet light. In other words, this suggests a possibility of the emergence of a severe change in the ecosystem some years after the destruction of the ozone layer.
[0006] As described above, it can be said that photoreactivation capable of reducing the influences of ultraviolet light using the energy of visible light supplied by sun in the same manner as for ultraviolet light is a considerably effective ultraviolet protective system for plants hardly capable of avoiding the influences of ultraviolet light in sunlight. A report showing the presence of photoreactivation in higher plants is issued, for supporting those described above.
[0007] A report suggests the presence of CPD photoreactivation activity in a higher plant "Oryza" particularly familiar to the Japanese. At the experiment, an appropriate dose of ultraviolet light irradiates the leaf (third leaf) of Oryza, which is subsequently irradiated with visible light (blue light). Then, the amount of CPD in the cells decreases (repairing) in proportion to the duration of visible light irradiation. Depending on the level of visible light irradiated on an individual after germination, additionally, the CPD repair efficiency of the individual was elevated (5). In other words, a larger amount of CPD can be repaired by the same dose of visible light. The CPD photoreactivation activity never similarly occurs in all of Oryza species. An Oryza species (Norin No.1) with poor ultraviolet resistance is repaired at a slow rate, compared with an Oryza species (Sasanishiki) resistant to ultraviolet light (6). This may possibly be ascribed to the occurrence of some mutation in the photoreactivating enzyme itself in the species with poor ultraviolet resistance or the system regulating the expression. However, the cause has not yet been elucidated.
[0008] References and information of the related art in relation with the invention of this application are as follows.
(1) Aziz Sancar. (1994) "Structure and Function of DNA photolyase" Biochemistr y 33:2-9.
(2) Takeshi Todo , Hiroshi Takemori , Haruko Ryo , Makoto Ihara , Tsukasa M atsunaga , Osamu Nikaido , Kenji Sato , Taisei Nomura (1993) "A new photore activation enzyme that specifically repairs ultraviolet light-induced (6-4) photopro duct " Nature 361:371-374.
(3) Aziz Sancar (1996) "No "End of History" for Photolyases " Science 272:48-4 9
(4) Gerhard Ries,Wemer Heller,Holger Puchta,Heninrich Sandermann,Harald K.Sei dlitz , Barbaara Hohn (2000) "Elevated UV-B radiation reduces genome stability in plants" Nature 406
(5) Hye-Sook Kang, Jun Hidema and Tadashi Kumagai (1998) "Effects of light environment during culture on UV-induced cyclobutyl pyrimidine dimers and thei r photorepair in rice (Oryza sativa L.)" Photochemistry and Photobiology 68: 71-77
(6) Jun Hidema, Tadashi Kumagai, John C. Sutherland, Betsy M Sutherland (199 7) "Ultraviolet B-sensitive rice cultivar deficient in cyclobutyl pyrimidine dimer r epair". Plant Physiology 113: 39-44
(7) Satoshi Nakajima, Munetaka Sugiyama, Shigenori Iwai, Kenichi Hitomi,Eriko Otoshi,Sang-Tae Kim,Cai-Zhong Jiang,Takishi Todo,Anne B Britt , Kazuo Yama moto (1998) "Cloning and characterization of a gene (UVR3) required for photor epair of 6-4 photoproducts in Arabidopsis thaliana" Nucleic Acid Research 26:63 8-644
(8) Jason L.Petersen,Darin W.Lang , Gary D.Small (1999) "Cloning and character ization of a class II DNA photolyase from Chlamydomonas" Plant Molecular Bio logy 40 :1063-1071
(9) Yao-Guang Liu, Kiyotaka Nagaki, Masao Fujita, Kanako Kawaura, Masahiko Uozumi, Yasunari Ogihara. (2000) "Development of an efficient maintenance an d screening system for large-insert genomic DNA libraries of hexaploid wheat in a transformation-competent artificial chromosome (TAC) vector." The Plant Jour nal 23 : 687-95.
(10) Kazuo Maruyama , Sumio Sugano (1994) "oligo-capping:a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides" Gene 138:171-174
(11) Michael Herrler (2000) "Use of SMART-generated cDNA for Differential G ene Expression Studies " Jounal of Molecular Medicine 78:B23.
Disclosure of the Invention
[0009] It is an object of the invention to obtain the information about the plant photoreactivation mechanism not so much elucidated yet, by isolating a photoreactivating enzyme from an Oryza species (Sasanishiki) resistant to ultraviolet light, where the presence of a photoreactivating enzyme is suggested as described above and to give a solution of environmental problems brought about by the destruction of the ozone layer and a solution of a serious problem in the 21 century, namely the food problem by preparing plants resistant to ultraviolet light using a cloned gene.
[0010] Accordingly, the invention relates to the following individual aspects.
1. A gene encoding a photoreactivating enzyme derived from Oryza.
2. The gene described in the aspect 1, where the gene encodes a pyrimidine dimer photoreactivating enzyme.
3. The gene described in the aspect 1 or 2, where the gene is derived from the species resistant to ultraviolet light.
4. The gene described in the aspect 1, 2 or 3, where the Oryza species is Sasanishiki.
5. The gene described in the aspect 1, where the Oryza species is Gulfmont.
6. A gene including a nucleotide sequence encoding the following protein (a) or (b):
(a) a protein comprising an amino acid sequence at the 174th position to 506th position from the N terminus of SEQ ID No.1;
(b) a protein comprising an amino acid sequence derived from the amino acid sequence (a) by deletion, substitution or addition of one to several amino acids, and having the activity of a photoreactivating enzyme.
7. A gene including the following DNA (a) or (b):
(a) DNA including base pairs at the 520th position to 1521st position in the nucleotide sequence of SEQ ID No.2;
(b) DNA hybridizing with the DNA comprising the nucleotide sequence (a) under stringent conditions and encoding a protein with the activity of a photoreactivating enzyme.
8. A gene encoding the following protein (a) or (b):
(a) a protein comprising the amino acid sequence of SEQ ID No.1;
(b) a protein comprising an amino acid sequence derived from the amino acid sequence (a) by deletion, substitution or addition of one to several amino acids, and having the activity of a photoreactivating enzyme.
9. A gene including the following DNA (a) or (b):
(a) DNA comprising the nucleotide sequence of SEQ ID No.2;
(b) DNA hybridizing with the DNA including the nucleotide sequence (a) under stringent conditions and encoding a protein with the activity of a photoreactivating enzyme.
10. A method for preparing a gene described in any one of the aspects 1 through 9, including a step of screening an Oryza gene library by repeating dilution PCR.
11. The method described in the aspect 10, where the Oryza gene library is a cDNA library.
12. The method described in the aspect 11, where Oryza is Sasanishiki or Gulfmont.
13. A recombinant expression vehicle containing a gene described in any one of the aspects 1 through 9.
14. The recombinant expression vehicle described in the aspect 13, where the recombinant expression vehicle is lamda phage.
15. The recombinant expression vehicle described in the aspect 13, where the recombinant expression vehicle is a plasmid vector.
16. A transformant prepared by transformation with an expression vehicle described in the aspect 13, 14 or 15.
17. The transformant described in the aspect 16, where the transformant is a plant.
18. The transformant described in the aspect 17, where the plant is Oryza.
19. The transformant described in the aspect 16, where the transformant is Escherichia coli.
20. A method for giving the resistance to ultraviolet light to a host or enhancing the resistance of a host against ultraviolet light, including a step of transforming the host with a gene described in any one of the aspects 1 through 9.
21. The method described in the aspect 20, where the host is a plant.
22. The method described in the aspect 21, where the plant is Oryza.
23. The method described in the aspect 22, where Oryza is of a species sensitive to ultraviolet light.
24. The method described in the aspect 23, where the species of Oryza sensitive to ultraviolet light is Norin No.1.
25. A method for screening the expression level of the gene of a photoreactivating enzyme in Oryza, using a gene described in any one of the aspects I through 9 or a DNA fragment thereof.
26. A method for assaying the transcription level of the gene of a photoreactivating enzyme to mRNA in Oryza by a Northern hybridization method using a gene described in any one of the aspects 1 through 9 or a DNA fragment thereof.
27. The method described in the aspect 25 or 26, where Oryza is of a species resistant to ultraviolet light and/or sensitive to ultraviolet light.
28. A polypeptide or protein encoded by a gene described in any one of the aspects 1 through 9.
29. The polypeptide or protein described in the aspect 28, where the polypeptide or protein has the activity of a photoreactivating enzyme.
30. A method for preparing a gene encoding a photoreactivating enzyme derived from a plant, including a step of preparing a first primer based on a highly homologous region in amino acid sequence between at least two types of existing photoreactivating enzymes, a step of cloning a first DNA fragment using the genome of the plant as template by PCR using the first primer, a step of preparing a second primer based on a highly homologous region in amino acid sequence between the first DNA fragment and the photoreactivating enzyme of Arabidopsis thaliana, a step of cloning a second DNA fragment using the plant gene library as template by PCR using the second primer, and a step of cloning the objective gene by a nucleic acid hybridization method using the second DNA fragment as a probe.
31. The method described in the aspect 30, where the existing photoreactivating enzymes are the photoreactivating enzymes of Arabidopsis thaliana and Chlorophyceae.
32. The method described in the aspect 30 or 31, where the plant gene library is a cDNA library.
33. The method described in the aspect 30, 31 or 32, where the nucleic acid hybridization method is a plaque hybridization method.
34. The method described in any one of the aspects 30 through 33, where the plant is Oryza.
35. The method described in the aspect 30, where the gene is a gene described in any one of the aspects 1 through 9.
Brief Description of the Drawings
[0011]
Fig.1 shows the photoreactivation mechanism. Ultraviolet light generates CPD or 6-4 adduct on the DNA strand (A). The CPD photoreactivating enzyme and the 6-4 adduct photoreactivating enzyme is bound specifically to the CPD and the 6-4 adduct, respectively (B), to bring back the individual damages to the original states under irradiation of visible light (near ultraviolet light, blue light) (C). Consequently, the photoreactivating enzymes are dissociated from the DNA strand (D).
Fig.2 shows the comparison in amino acid sequence of photoreactivating enzyme between Arabidopsis thaliana and Chlorophyceae. A.t expresses Arabidopsis thaliana and C.r expresses Chlorophyceae, while AC 1 to AC5 express primer positions.
Fig.3 shows a schematic view of screening the Oryza Gulfmont cDNA/pSPORT-T library and the Sasanishiki cDNA/pBSSK(-) library.
Fig.4 shows an evolution tree for photoreactivating enzymes, blue color receptors and the circadian rhythm receptor family.
Fig. 5 shows the amplification of the coding region of the CPD photoreactivating enzyme in the Oryza genome with mix primers.
Fig.6 shows the comparison between the amino acid sequence speculated from the Oryza genome AC1.1 kbp and the amino acid sequence of the CPD photoreactivating enzyme in Arabidopsis thaliana. Herein, portions with double underlines show the positions of perfect match primers.
Fig.7 shows the comparison in amino acid sequence between a CPD photoreactivating enzyme fragment in the Oryza genome and the CPD photoreactivating enzyme in Arabidopsis thaliana. Herein, portions with double underlines show the positions of perfect match primers.
Fig.8 shows the comparison in amino acid sequence between a CPD photoreactivating enzyme fragment in the Oryza (Gulfmont) and the CPD photoreactivating enzyme in Arabidopsis thaliana.
Fig.9 shows the results of a complementarity test verifying that the photoreactivating enzyme of the invention has photoreactivation ability, using an Escherichia coli strain deficient in repairing.
Fig. 10 is a schematic view showing the procedures for giving resistance to ultraviolet light to a species ultraviolet-sensitive or allowing a strain resistant to ultraviolet light to more highly express the CPD photoreactivating enzyme, through the introduction of the gene of the invention.
Best Mode for Carrying out the Invention
[0012] As shown in the following Examples, the individual genes encoding the photoreactivating enzyme derived from Oryza in accordance with the invention was prepared, using for example the cDNA libraries derived from "Sasanishiki" as an Oryza species with resistance to ultraviolet light as disclosed in the specification and "Gulfmont", by PCR using appropriate primers prepared in accordance with the invention, particularly the primers prepared on the basis of the amino acid sequence highly conserved between the pyrimidine dimer (CPD) photoreactivating enzymes from Arabidopsis thaliana and Chlorophyceae. By repetition of the dilution PCR, in particular, gene libraries such as the cDNA libraries of Oryza were screened to recover the gene of the invention efficiently.
[0013] The invention relates to a gene containing a nucleotide sequence encoding the protein comprising the amino acid sequence from the 174th position to 506th position from the N terminus of SEQ ID No.1, particularly a gene with an additional appropriate nucleotide sequence on the 5' side. The sequence is preferably derived from Oryza, for example "Sasanishiki". One example thereof includes a gene encoding the protein of the amino acid sequence represented by SEQ ID No.1.
[0014] Based on the sequence information disclosed in the specification, a person skilled in the art can prepare such gene by chemical synthesis and the like using well known techniques in the art. Further, a person skilled in the art can readily carry out the deletion, substitution or addition of one to several amino acids in a specified amino acid sequence in SEQ ID No. 1 by using well known techniques in the art, so that the photoreactivating enzyme activity may be substantially conserved.
[0015] The specific example of the above-described gene includes a gene containing DNA including base pairs at the 520th position to 1521st position in the nucleotide sequence of SEQ ID No.2, particularly a gene containing DNA of the nucleotide sequence of SEQ ID No.2.
[0016] In accordance with the invention, a specific gene or DNA can be hybridized in a buffer solution well known to a person skilled in the art under stringent conditions of various conditions such as appropriate temperature and salt concentration. The DNA being hybridizable to the gene or DNA of the invention under such stringent conditions and still having an activity substantially equivalent to the photoreactivating enzyme includes for example DNA with homology of 90 % or more, preferably 95 % or more, more preferably 98 % or more and furthermore preferably more than 99.5 % to each of the corresponding genes.
[0017] Further, in another embodiment, further, the invention relates to a recombinant expression vehicle carrying at least one gene of the gene in accordance with the invention. The recombinant expression vehicle includes appropriate ones known to a person skilled in the art, such as various vectors. Particularly, various plasmid vectors such as Ti plasmid contained in bacteria of the genus Agrobacterium and phage vectors such as lamda phage are preferable.
[0018] The recombinant expression vehicle may include various sequences known to a person skilled in the art for gene recombinant manipulation, for example various promoters as binding domains for various σ subunits as transcription factors in prokaryotic cells such as Escherichia coli, and various transcription regulatory elements such as enhancer, restriction enzyme sites, as well as genes of selection markers (marker enzymes, etc.) such as kanamycin resistant marker and the recombinant expression vehicle can be readily prepared by methods known to a person skilled in the art.
[0019] The invention further relates to a host, transformed by the above expression vehicle, particularly a plant such as Oryza.
[0020] The transformation of a host with the gene of the invention can give or enhance the resistance to ultraviolet light in the host. The host is preferably plants for example Oryza, particularly an Oryza species sensitive to ultraviolet light, such as "Norin No.1".
[0021] Using the gene of the invention or a DNA fragment thereof as, for example, a probe, the expression level of a photoreactivating enzyme gene in plants such as Oryza can be screened. This can be done by assaying the transcription level of the photoreactivating enzyme gene to mRNA in Oryza by the Northern hybridization method using the gene of the invention or a DNA fragment thereof as a probe.
[0022] Further, the invention relates to a polypeptide or protein with a photoreactivating enzyme activity, as encoded by the gene of the invention. The polypeptide or protein can be prepared by culturing the transformant and using the resulting culture supernatant or the resulting bacterial cell. As to the culture conditions and the separation and purification from the culture supernatant, a person skilled in the art can appropriately select such conditions and the like with reference to the Examples in this specification.
[0023] The invention further relates to a method for preparing a gene encoding a photoreactivating enzyme derived from a plant, including a step of preparing a first primer based on a highly homologous region in amino acid sequence between at least two types of existing photoreactivating enzymes, a step of cloning a first DNA fragment using the genome of the plant as template by PCR using the first primer, a step of preparing a second primer based on a highly homologous region in amino acid sequence between the first DNA fragment and the photoreactivating enzyme of Arabidopsis thaliana, a step of cloning a second DNA fragment using the plant gene library as template by PCR using the second primer, and a step of cloning the objective gene by a nucleic acid hybridization method using the second DNA fragment as a probe. The existing photoreactivating enzymes are the photoreactivating enzymes of Arabidopsis thaliana and Chlorophyceae. There is no limitation as to the source used for recovering a sample used for preparing the plant gene library, however, cDNA library is preferable. The nucleic acid hybridization method includes any method known to a person skilled in the art, for example plaque hybridization method and Southern hybridization method. Further, the plant includes for example Oryza, wheat, and barley. By such method, the gene of the invention can be prepared.
[0024] The contents described in the Japanese Patent Application 2001-320138 are all included in this specification.
Examples
[0025] The invention is now specifically described in the following Examples. However, the invention is not limited to these Examples.
Materials and Methods
1. Escherichia coli and plasmids
[0026] The Escherichia coli used was XL1-Blue (Δ(lac), endA1, gyrA96, hsdR17 (rk -,mk+), recA1, relA1, supE44, thi-1, [F',lac1q, lacZΔM15, proAB, Tn 10 (tetr)]) N K J 3 0 0 2 (Δ(lac-proAB), endA1, gyrA96, hsdR17 (rk-,mk+), relA1, supE44, thi-1, phr20::Kan uvrA::Kan ΔrecA, [F',lac1q, lacZΔM15, proAB]). pGEM-T and pGEM-T easy vector (Promega) were used for the cloning of PCR products. So as to examine the photoreactivation ability of the cloned CPD photoreactivating enzyme gene from Sasanishiki, the gene was integrated in the pTZ18R vector (PHARMACIA) and then introduced in Escherichia coli.
2. Preparation of cDNA library of Oryza Sasanishiki
[0027] The Oryza species was grown in the environment under the irradiation of visible light since the germination of Oryza seed until the third to sixth leaves opened. Then, mRNA was extracted from the individual leaves. Using mRNA as template, cDNA was synthetically prepared. An adapter was ligated to both ends of the cDNA and was then packaged in Lamda ZAP II (STRATAGENE), to prepare the library.
3. Preparation and sequencing of mix primers
[0028] Based on the amino acid sequences at five positions (Fig.2; AC1 to AC5) where the amino acid sequences were highly conserved between the CPD photoreactivating enzymes from Arabidopsis thaliana (7) and Chlorophyceae (8), mix primers were prepared. Using about 1 µg of the Oryza genome as template, PCR was conducted ([Mg2+) = 2.0 mM; 93 °C for 1 min; 40 cycles of << 93 °C for 1 min, 53 °C for 1 min and 72 °C for 1.5 min>>; 72 °C for 10 min.: Gene Amp 480/9600 System by Perkin Elmer). The PCR product was cloned in pGEM-T or the pGEM-T easy vector (Promega), and its nucleotide sequence was determined via fluorescent labeling using ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kits or the DYEnamic ET terminator Cycle Sequencing pre-mix kit by 310 Genetic Analyzer or 373 Genetic Analyzer (Perkin Elmer).
4. Screening of Lamda ZAP II/rice cDNA library by plaque hybridization
[0029] About 15,000 to 20,000 plaques of Lamda phage (Lamda ZAP II/rice cDNA library) were grown on a culture medium and then covered with sterilized Gene Screen plus (NEN Life Science Products) for transfer. The transferred Gene Screen plus was immersed in a denature solution (0.5N NaOH, 1.5M NaCl) for 5 minutes, and continuously immersed in a nautring solution (0.5M Tris-HCl, 1.5M NaCl) for 5 minutes and then in 2 x SSC solution for 5 minutes, and spontaneously dried for 30 to 60 minutes, followed by baking in a dry heat sterilizer at 80 °C for 2 hours. This was treated with 3 × SSC (65 °C) for 30 minutes and a pre-hybridization solution (5× SSC, 1 % SDS, 1 × Denhart: 65 °C) for 2 hours, and mixed about 25 ng of a 32P-labeled probe in a hybridization solution (0.75 M NaCl, 20 mM Tris-HCl, pH 8.0, 2.5 mM EDTA, pH 8.0, 1 % SDS, 1× Denhart, 10 µg/ml sermon sperm DNA). The resulting mixture was kept overnight at 65 °C (hybridization). The Gene Screen plus after overnight hybridization was taken out and rinsed in a solution of 2 × SSC and 0.1 % SDS for 5 minutes and twice in a solution of 0.2 × SSC and 0.1 % SDS (65 °C) for 30 minutes. Then, the resulting product was covered with FUJI MEDICAL X-ray FILM (FUJI FILM) for exposure and development. A plaque with developed signals was recovered from the culture medium. A plaque was formed on a fresh culture medium and for confirmation, the plaque hybridization was again carried out by the same procedures. The plaque was transferred on pBlueScript SK(-) for the determination of the nucleotide sequence of the insert.
5. Genome Walking
[0030] About 5 µg of the genome extracted from the Oryza was treated with four types of restriction enzymes (Dra I, EcoR V, Stu I, Pvu II), for adapter ligation (see Universal Genome Walker Kit Manual manufactured by CLONTECH). Using the resulting ligate as template, PCR was conducted using the adapter primer and the primers prepared on the basis of the cDNA sequence of Oryza as already determined ([Mg2+] = 2.0 mM; 93 °C for I min; 40 cycles of << 93 °C for 1 min, 50-55 °C for 1 min and 72 °C for 1.5 min>>; 72 °C for 10 min.: Gene Amp 480/9600 System by Perkin Elmer). The resulting PCR product was applied to electrophoresis and then transferred on Gene Screen plus for Southern hybridization, to screen the intended bands. Again, the PCR product was developed by electrophoresis on low melting agar (LMA), to extract a band with signals, which was then cloned into pGEM-T or the pGEM-T easy vector (Promega) for sequencing.
6. Screening the Oryza Gulfmont cDNA/pSPORT-T library or the Sasanishiki cDNA/pBS SK(-)/lamda Zap II library
[0031] The screening of SUPERSCRIPT RICE (cv. Gulfmont) LEAF cDNA library and the manufactured by GIBCO BRL Sasanishiki cDNA/pBS SK(-) library prepared by transferring the Lamda ZAP II/rice cDNA library in pBS SK(-) plasmid was conducted by the dilution PCR ([Mg2+] = 1.0 mM; 93 °C for 1 min; 40 cycles of << 93 °C for 1 min, 60 °C for 1 min and 72 °C for 1.5 min>>; 72 °C for 10 min.: Gene Amp 480/9600 System by Perkin Elmer). So as to determine the ratio of the target plasmid (the cDNA of the CPD photoreactivating enzyme) at the state of stock solution (5 x 109 Escherichia coli cells/ml), the stock solution was diluted by every 10-1 order to 10-2, 10-3, 10-4, 10-5, 10-6, and 10-7 in each volume of 5 ml. Then, the diluted solutions were cultured up to the stationary phase for amplification, from which the plasmid was extracted for PCR. 5 ml of the 10-6 -fold dilution solution did not contain the target, while 5 ml of the 10-5 -fold dilution solution contained the targets 1 to 9. A liquid culture of 5 ml of a solution at the most diluted concentration of 10-5 involving signal generation was again prepared and divided into 10 portions (#1 to 10), which were individually cultured up to the stationary phase for amplification. Then, the plasmid was extracted for PCR (Fig. 3). #1 and #3 contain the target at a concentration 10-fold higher than the concentration in the stock solution. The liquid culture involving signal generation was treated by the same procedures as for the stock solution in a repeated manner, to target just one colony.
Results and Discussion
(1) Preparation of mix primers
[0032] Currently, CPD photoreactivating enzymes have been isolated from various biological organisms. They are homologous to the blue receptors in plants and the genes responsible for human circadian rhythm, and form one family (Fig. 4). Based on the difference in amino acid sequence, they are broadly divided into two groups. With some exception, the two groups are Class I to which microorganisms such as Escherichia coli belongs and Class II to which higher eukaryotic organisms belong. Among plants, Arabidopsis thaliana and Chlorophyceae with isolated CPD photoreactivating enzymes belong to Class II. Because Oryza as a current experimental subject is a higher eukaryotic organism, it is assumed that the amino acid sequence of the photoreactivating enzyme thereof may belong to those of Class II, like Arabidopsis thaliana and Chlorophyceae. On the comparison in amino acid sequence between Arabidopsis thaliana and Chlorophyceae, primers were synthetically prepared on the basis of the five regions with higher homology (Fig. 2). As several types of codons correspond to one amino acid determination, the primers are mix primers containing all the nucleotide sequences corresponding to the amino acid sequences at the individual regions. If it is confirmed by PCR for all possible combinations of the five primers that the primary structure of the presently used photoreactivating enzyme of Oryza contains the same amino acid sequence as the original amino acid sequence working for the primer preparation, it suggests that the genome DNA includes the presence of a nucleotide sequence corresponding thereto. In that case, theoretically, the nucleotide sequence in the sequence can be amplified and determined by PCR.
(2) PCR using Oryza genome DNA as template
[0033] Fig.5 shows actual PCR with the primers AC1 to AC5 described above, using the Oryza genome DNA as template. AC 1-2 and the like show primer combinations; the AC 1-2 shows the coding region of the CPD photoreactivating enzyme of the Oryza genome in the primers AC1 and AC2. Consequently, a combination of AC2 and AC4 gave a DNA fragment of about 1.1 kbp, while a combination of AC3 and AC4 gave a DNA fragment of about 600 bp. As shown in the figure, the 600-bp fragment is not amplified at a level as high as the level for the 1.1-kbp fragment. AC 2-4 includes 245 amino acids (735 bp) in Arabidopsis thaliana or 244 amino acids (732 bp) in Chlorophyceae. AC 3-4 includes 127 amino acids (381 bp) in Arabidopsis thaliana and also 127 amino acids (381 bp) in Chlorophyceae. It is estimated that because an intron may exist in the genome DNA's, the sequence will be longer than this. Thus, the fragments of 1.1 kbp and 600 bp might be the intended fragments. Even under modified temperature condition and Mg2+ concentration, totally no PCR product was obtained with other primers. The PCR product was cloned into the pGEM-T vector (CLONTECH), to determine the sequences of the 1.1-kbp fragment of AC 2-4 and the 600-bp fragment of AC 3-4 to speculate their amino acid sequences. Its comparison with the amino acid sequence of the CPD photoreactivating enzyme of Arabidopsis thaliana shows high homology to the amino acid sequence from the 1.1-kbp DNA sequence (Fig. 6). Because the 600-bp fragment was not so much amplified by PCR as described above, it is considered because the primer might have annealed to a sequence similar to the original amino acid sequence and then amplified, the fragment might have been a background.
(3) Screening of Oryza cDNA library
[0034] So as to obtain a cDNA fragment without intron, a primer was prepared from a region with high homology to the CPD photoreactivating enzyme of Arabidopsis thaliana (the underlined part in Fig.6) in the 1.1-kbp sequence. Using the primer, and using the Oryza cDNA as a template, PCR was conducted (93 °C for 1 min; 40 cycles of<< 93 °C for 1 min, 55 °C for 1 min and 72 °C for 1.5 min>>; 72 °C for 10 min.: Gene Amp System by Perkin Elmer)), to obtain a 600-bp fragment. In the same manner as in the case of the genome DNA, the sequence was determined to speculate the amino acids, which was compared with that of the CPD reactivation enzyme of Arabidopsis thaliana. It was confirmed that these amino acids were highly homologous at the same positions as that of the 1.1-kbp fragment of the genome DNA. Further, using the cDNA fragment as a probe, the Lamda ZAP II/Rice cDNA library as the starting material for the 600-bp template was used for plaque formation, for screening by Southern hybridization several times. About 200,000 plaques were screened, so that two candidates were obtained. The nucleotide sequence of DNA packaged in each of the two plaques was determined. It was shown that both the plaques had the same sequence, which was a 1.1-kbp cDNA fragment including the 600-bp probe partially deficient in a sequence at the 5' side up to the 3' terminus (C terminus) (Fig. 7). The deficiency at the 5' side may be ascribed to the decomposition of the probe from the 5' side with RNase attached on the device and the like, before cDNA formation by the reverse-transcription of mRNA as the template for the cDNA. Because the decomposition is not uniform, the cDNA library contains fragments of various lengths deficient in a sequence at the 5' side in addition to the full-length fragment. This is the case with the 1.1-kbp fragment obtained by the plaque hybridization. A possibility remains that a fragment of a length closer to the full length may be obtained by carrying out the same experiment again. However, such fragment could not be obtained by the several experiments using the library.
(4) Genome Walking Method of Oryza genome DNA
[0035] Because the approach from the cDNA library was difficult as described above, the genome walking method was carried out for hardly readily decomposed genome DNA unlike cDNA. As described in the "Materials and Methods", Oryza genome DNA was treated by four restriction enzyme types (Dra I, EcoR V, Stu I, Pvu II), and an adapter was ligated to the resulting digestion products (Genome Walker Library: CLONTECH). Using the resulting products as a template, PCR with a primer for the ligated adapter and the prepared perfect match primers was conducted. Consequently, plural DNA fragments were obtained by PCR using the library treated with Pvu II as template and GSP4 and the primer for the adapter. Then, by Southern hybridization, the intended fragment was narrowed down around 500 bp among the resulting plural PCR products, cloned and determine the sequences. Consequently, a region of only 100 bp at the 5' side was confirmed, which was unknown (no homology to other CPD photoreactivating enzymes could be observed even after the conversion to amino acid sequence). Libraries except for the Pvu II library could not be well screened by Southern hybridization. Thus, not any more effect could be obtained.
(5) Screening of different Oryza cDNA library
[0036] As described above, only fragments with the deficiency at the 5' side were obtained from the Oryza (Sasanishiki) Lamda ZAP II/cDNA library. Accordingly, the present inventors screened the pSPORT-T/rice (Gulfmont) cDNA library prepared via another route. The Gulfmont species is a species produced in USA and its photoreactivation activity has not yet been examined. Screening was conducted using as a marker the amplification of a known fragment by the dilution PCR (Fig. 3). First, a 10-1 dilution series of the stock solution of the library is cultured, from which the plasmid is extracted for PCR. A liquid culture with a positive band at the lowest concentration is prepared in the same manner. The total volume of the dilution solution is divided into equal 10 portions, for culturing. In such manner, some of 10 test tubes contain the intended plasmid; while the remaining test tubes do not contain the plasmid. Because a 10-fold dilution of the liquid culture divided in such 10 portions has no positive band, theoretically, all the test tubes are never positive. The plasmid is extracted from the individual 10 test tubes, for PCR. A test tube with a positive band contains the target gene at a higher level than the level in the stock solution, because other portions with no content of the plasmid are preliminarily removed from the test tube. This procedure is repeatedly carried out to concentrate the target, so that only one plasmid can be screened. In such manner, a plasmid containing an insert of about 1.8 kbp was obtained. The nucleotide sequence of the fragment was determined. As a result of comparing with the cDNA sequence of Sasanishiki insofar determined, 99.5 % homology was revealed. However, when the sequence was converted to amino acid sequence, it was shown that the sequence included a sequence of a 4-bp base inserted at a position in the sequence of Sasanishiki, which indicates that a frame shift occurred at the position. Excluding the position, the nucleotide sequence was converted to amino acid sequence. Then, it was shown that the resulting amino acid sequence had high homology to the amino acid sequence of the CPD photoreactivating enzyme of Arabidopsis thaliana (Fig. 8).
[0037] Using the Sasanishiki cDNA/pBS SK(-) library prepared from the Lamda ZAP II/rice cDNA library as template, further, screening was conducted by the above dilution PCR, for the cloning of a gene encoding a photoreactivating enzyme from Sasanishiki of the photoreactivation wild type. The resulting nucleotide sequence is shown as SEQ ID NO.2 (1521 base pairs), while the amino acid sequence of the protein encoded by the base pairs is shown as SEQ ID NO. 1 (the number of amino acid residues: 506).
(6) Verification of photoreactivation ability of CPD photoreactivating enzyme gene
[0038] So as to further verify the photoreactivation ability of the CPD photoreactivating enzyme gene of Sasanishiki as recovered in accordance with the invention, additionally, the following experiment was carried out. First, NKJ3002 which is a deficient strain in all of the DNA repair abilities of Escherichia coli was prepared, followed by ultraviolet irradiation. Because NKJ3002 cannot repair DNA lesions such as CPD and 6-4 adduct generated by ultraviolet irradiation, the increase of the dose involves the decrease of the survival rate. Further, as the strain is also deficient in the photoreactivation gene of Escherichia coli, the irradiation of visible light after ultraviolet irradiation cannot cause any change, compared with no irradiation of visible light. A plasmid prepared for the expression of the CPD photoreactivating enzyme gene of Sasanishiki was introduced into the strain, for carrying out the same experiment. Consequently, it was observed that the survival rate was improved distinctly by the irradiation of visible light after ultraviolet irradiation. The CPD photoreactivating enzyme gene of Sasanishiki compensated the ultraviolet sensitivity of the photoreactivation gene-deficient Escherichia coli. In other words, it was absolutely verified that the product of the CPD photoreactivating enzyme gene of Sasanishiki (the photoreactivating enzyme in accordance with the invention) should absolutely have the photoreactivation ability.
Industrial Applicability
[0039] As described above, Oryza includes ultraviolet resistant species (Sasanishiki) and ultraviolet sensitive species (Norm No.1) (6). The difference in photoreactivation activity between these two species considerably close to each other in terms of strain has not yet been elucidated. However, the Northern hybridization using the novel gene obtained in accordance with the invention and the cDNA fragment thereof enables the comparison in mRNA transcription level (enzyme expression level) between the CPD photoreactivating enzymes of the two species. Additionally, visible light irradiated after germination (leaf development) promotes the photoreactivation activity of Oryza. The activity reaches the maximum when the leaves completely develop. The induction of photoreactivation activity in photoenvironment differs depending on the plant. In case of Oryza, visible light elevates the induction as described above, while in case of Arabidopsis thaliana, even ultraviolet light can induce the activity. As described above, the induction of the photoreactivation activity of the plants in photoenvironment is largely not yet elucidated. It is expected that the Northern hybridization using the gene obtained at the present time and the cDNA fragment thereof in various photoenvironment at various growth stages will provide a clue for the elucidation of what kind of influences different photo-environment makes to the individual growth stages of Oryza.
[0040] The gene obtained in accordance with the invention can be used further to carry out the complementarity test of Escherichia coli of a deficient type in photoreactivating enzyme, the crystallization of the enzyme, and the preparation of an antibody. Additionally, the distribution of a CPD photoreactivating enzyme in a plant cell can be observed by in situ hybridization with the antibody, to elucidate the involvement of the enzyme in DNA (mitochondria, chlorophyll) other than nucleus. By the gene introduction technology, additionally, ultraviolet resistance can be given to an ultraviolet sensitive species (for example, Norin No.1); the complementarity test can be carried out; or a species at the higher expression level of a CPD photoreactivating enzyme can be generated from an ultraviolet resistant strain (Fig. 10). The species at the higher level of the CPD photoreactivating enzyme will potentially be produced in a stable manner in the environment of an enormous dose of ultraviolet light due to a possible destruction of the ozone layer in future.
(a) a protein comprising an amino acid sequence at the 174th position to 506th position from the N terminus of SEQ ID No.1;
(b) a protein comprising an amino acid sequence derived from the amino acid sequence (a) by deletion, substitution or addition of one to several amino acids, and having the activity of a photoreactivating enzyme.
(a) DNA including base pairs at the 520th position to 1521st position in the nucleotide sequence of SEQ ID No.2;
(b) DNA hybridizing with the DNA comprising the nucleotide sequence (a) under stringent conditions and encoding a protein with the activity of a photoreactivating enzyme.
(a) a protein comprising the amino acid sequence of SEQ ID No.1;
(b) a protein comprising an amino acid sequence derived from the amino acid sequence (a) by deletion, substitution or addition of one to several amino acids, and having the activity of a photoreactivating enzyme.
(a) DNA comprising the nucleotide sequence of SEQ ID No.2;
(b) DNA hybridizing with the DNA including the nucleotide sequence (a) under stringent conditions and encoding a protein with the activity of a photoreactivating enzyme.